Researchers at the University of Tokyo have found a way to increase the sensitivity of existing quantitative phase imaging so that all structures in living cells can be seen simultaneously, from small particles to large structures. This artistic representation of the technique shows pulses of sculpted light (green, top) traveling through a cell (center) and going out (bottom) where changes in the light waves can be analyzed and converted into a more detailed image. Credit: s-graphics.co.jp, CC BY-NC-ND
Upgrade to quantitative phase imaging can increase image clarity by increasing dynamic range.
Experts in optical physics have developed a new way to look inside living cells in greater detail using existing microscopy technology and without the need to add stains or fluorescent dyes.
Because individual cells are nearly translucent, microscope cameras must detect extremely subtle differences in the light passing through parts of the cell. Those differences are known as the phase of light. Camera image sensors are limited by the amount of light phase difference they can detect, also known as dynamic range.
“To see more details with the same image sensor, we need to increase the dynamic range so that we can detect smaller phase changes of light,” said associate professor Takuro Ideguchi of the University of Tokyo Institute for Photon Science and Technology.
The research team developed a technique to take two exposures to measure large and small changes in the light phase separately and then seamlessly connect them to create a highly detailed final image. They called their method adaptive dynamic range shift quantitative phase imaging (ADRIFT-QPI) and recently published their results in Light: Science and Applications.
Images of silica beads taken using conventional quantitative phase imaging (top) and a clearer image produced using a new ADRIFT-QPI microscopy method (bottom) developed by a research team at the University of Tokyo. The photos on the left are images of the optical phase and the images on the right show the optical phase change due to the mid-infrared (molecular specific) light absorption by the silica grains. In this proof-of-concept demonstration, researchers calculated that they achieved about 7 times greater sensitivity with ADRIFT-QPI than those with conventional QPI. Credit: Image by Toda et al., CC-BY 4.0
“Our ADRIFT-QPI method does not require a special laser, no special microscope or image sensors; we can use living cells, we don’t need staining or fluorescence and the potential for phototoxicity is very small, ”said Ideguchi.
Phototoxicity refers to the killing of cells with light, which can become a problem with some other imaging techniques, such as fluorescence imaging.
Quantitative phase imaging sends a pulse of a flat sheet of light to the cell and then measures the phase shift of the light waves after they pass through the cell. Computer analysis then reconstructs an image of the main structures in the cell. Ideguchi and colleagues have previously developed other methods to improve quantitative phase microscopy.
Quantitative phase imaging is a powerful tool for examining individual cells because it allows researchers to make detailed measurements, such as tracking the growth rate of a cell based on the shift in light waves. However, the quantitative aspect of the technique has a low sensitivity due to the low saturation capacity of the image sensor, so tracking nanosized particles in and around cells is not possible with a conventional approach.
A standard image (top) created with conventional quantitative phase imaging and a clearer image (bottom) produced using a new ADRIFT-QPI microscopy method developed by a research team from the University of Tokyo. The pictures on the left are images of the optical phase and the images on the right show the optical phase change due to the mid-infrared (molecular specific) light absorption, mainly by proteins. Blue arrow points to the edge of the nucleus, white arrow points to the nucleoli (a substructure in the nucleus) and green arrows point to other large particles. Credit: Image by Toda et al., CC-BY 4.0
The new ADRIFT-QPI method has overcome the limitation of the dynamic range of quantitative phase imaging. During ADRIFT-QPI, the camera takes two exposures and produces a final image that is seven times more sensitive than traditional quantitative phase microscopy images.
The first exposure is produced with conventional quantitative phase imaging – a flat sheet of light is pulsed towards the sample and the phase shifts of the light are measured after it passes through the sample. A computer image analysis program develops an image of the sample based on the initial exposure and then quickly designs a sculpted wavefront of light that reflects that image of the sample. A separate component called a wavefront shaping device then generates this “light sculpture” with higher intensity light for stronger illumination and pulses it to the sample for a second exposure.
If the first exposure produced an image that was a perfect representation of the sample, the tailored light waves from the second exposure would penetrate the sample at several stages, pass through the sample, and then emerge as a flat sheet of light, exposing the camera to see nothing but a dark image.
“Here’s the interesting thing: we’re erasing the image of the monster a bit. We hardly want to see anything. We are scrapping the big structures so we can see the smaller ones in detail, ”explained Ideguchi.
In reality, the first exposure is not perfect, so the sculpted light waves emerge with subtle phase deviations.
The second image shows minute phase differences in light that were “washed out” by larger differences in the first image. This remaining small phase difference in light can be measured with increased sensitivity due to the stronger illumination used in the second shot.
Additional computer analysis reconstructs a final image of the sample with an expanded dynamic range of the two measurement results. In proof-of-concept demonstrations, researchers estimate that the ADRIFT-QPI produces images with seven times greater sensitivity than conventional quantitative phase imaging.
Ideguchi says the real advantage of ADRIFT-QPI is the ability to see tiny particles in the context of the whole living cell without needing labels or staining.
“For example, tiny signals from nanoscale particles, such as viruses or particles moving inside and outside a cell, can be detected, allowing simultaneous observation of their behavior and the state of the cell,” said Ideguchi.
Reference: “Adaptive Dynamic Range Shift (ADRIFT) Quantitative Phase Imaging” by K. Toda, M. Tamamitsu and T. Ideguchi, December 31, 2020, Light: Science and Applications.
DOI: 10.1038 / s41377-020-00435-z
Funding: Japan Science and Technology Agency, Japan Society for the Promotion of Science.